Nonpeptide Angiotensin II Receptor Antagonists: Synthesis, Biological

J. Med. Chem. 1996, 39, 323-338

323

Nonpeptide Angiotensin II Receptor Antagonists: Synthesis, Biological Activities, and Structure-Activity Relationships of Imidazole-5-carboxylic Acids Bearing Alkyl, Alkenyl, and Hydroxyalkyl Substituents at the 4-Position and Their Related Compounds Hiroaki Yanagisawa,* Yoshiya Amemiya, Takuro Kanazaki, Yasuo Shimoji, Koichi Fujimoto, Yoshiko Kitahara, Toshio Sada, Makoto Mizuno, Masahiro Ikeda, Shuichi Miyamoto, Youji Furukawa, and Hiroyuki Koike Research Institute, Sankyo Company, Ltd., 2-58, Hiromachi 1-chome, Shinagawa-ku, Tokyo 140, Japan Received June 19, 1995X

A series of imidazole-5-carboxylic acids bearing alkyl, alkenyl, and hydroxyalkyl substituents at the 4-position and their related compounds were prepared and evaluated for their antagonistic activities to the angiotensin II (AII) receptor. Among them, the 4-(1-hydroxyalkyl)imidazole derivatives had strong binding affinity to the AII receptor and potently inhibited the AII-induced pressor response by intravenous administration. Various esters of these acids showed potent and long-lasting antagonistic activity by oral administration. The most promising compounds were (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl (CS-866) and (pivaloyloxy)methyl esters of 4-(1-hydroxy-1-methylethyl)-2-propyl-1-[(2′-1H-tetrazol-5-ylbiphenyl-4-yl)methyl]imidazole-5-carboxylic acid (26c). A study involving stereochemical comparison of 26c with the acetylated C-terminal pentapeptide of AII was also undertaken. Introduction The great success of angiotensin-converting enzyme (ACE) inhibitors:1 captopril, enalapril, and others, which inhibit the formation of angiotensin II (AII) from angiotensin I (AI) in the renin-angiotensin system (RAS),2 in treatment of hypertension and congestive heart failure, led to recognition of the important role of the RAS in homeostasis of the cardiovascular system. Agents, that block the RAS at steps other than the one blocked by ACE inhibitors have also been studied during the last few decades. They have been expected to lack adverse effects3 observed with the use of ACE inhibitors, such as dry cough and angioedema, caused by the potentiation of bradykinin, substance P, and other biologically active peptides.4 Renin inhibitors,5 which interrupt the formation of AI, showed highly specific and potent inhibitory activity for human renin, but they have not yet been used in the clinical stage because of poor bioavailability and metabolic instability. On the other hand, AII receptor antagonists,6 which suppress the final stage of the RAS, look promising. AII analogue peptides,7 such as saralasin8 and sarmesin,9 are known to be potent antagonists and to lower blood pressure in humans, when administered intravenously.10 However, these peptides have been limited in clinical use because of ineffectiveness orally, metabolic instability, and their partial agonist activity.11 The first nonpeptide AII antagonists, 1-benzylimidazole-5-acetic acid derivatives, were reported by the Takeda group.12 Unfortunately, they did not have sufficient potency for clinical purposes. The DuPont group pursued a study in this field and developed losartan (DuP 753), 1, 13 which is orally effective and metabolized to the more potent full antagonist EXP 3174, 2.14 Since then, numerous new antagonists have been reported by various research groups.15 Losartan (1), now used in the clinical stage, and other antagonists in clinical trials selectively block the AT1 receptor, which is one of the two major subtypes X

Abstract published in Advance ACS Abstracts, November 1, 1995.

0022-2623/96/1839-0323$12.00/0

of the AII receptor, AT1 and AT2, and is responsible for most of the presently known pharmacological functions.16 Structure-activity relationships (SAR) of the imidazole antagonists have been reported.13c,17 Lipophilic substituents, like a biphenylylmethyl group at the 1-position and a linear alkyl group at the 2-position, which will associate with hydrophobic pockets of the receptor, and an acidic group, like tetrazole, CO2H, or NHSO2CF3, on the biphenylyl group at the 1-position, which will bind to a basic position of the receptor, are required for potent antagonistic activity. Furthermore, a nitrogen atom at the 3-position enhances the activity through hydrogen bonding to the receptor.18 However, the functions of substituents at the 4- and 5-positions on the imidazole ring in relation to the antagonistic activity had remained ambiguous at the time starting our study. The DuPont group13c recommended a lipophilic and electron-withdrawing group, such as iodine or CF3, as a substituent at the 4-position and a smallsized group, such as CH2OH, CH2OMe, or CO2H, which is capable of forming a hydrogen bond, as a substituent at the 5-position. Later, they reported on the SAR of alkyl- and (perfluoroalkyl)imidazoles17c,e and found that the imidazoles, which have ethyl and pentafluoroethyl groups at the 4-position, have highly antihypertensive potencies upon oral administration. On the other hand, the antagonist SK&F108566 (3),19 developed by SmithKline Beecham, has a large substituent, thienylmethyl acrylate group, at the 5-position, which mimics Phe8 of AII. Similarly, the 1,2,4-triazole antagonist 4, bearing a (2-carboxybenzyl)thio group at the 5-position, had © 1996 American Chemical Society

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potent binding affinity.20 These results suggested that the receptor would have a hydrophobic pocket and an ionic or a hydrogen-bonding site corresponding to the places near the 5-position of the imidazole nuclei. On the contrary, Thomas et al.18 suggested that chlorine and CH2OH in 1 would not be critical to binding, since the benzimidazole 5 had an antagonistic potency comparable to 1.

Molecular modeling and stereochemical comparison studies on imidazole antagonists and AII have been discussed by a few groups in order to understand a role of functional groups in antagonists.17a,19 The first attempt was done by the DuPont group.17a They overlayed the imidazole ring and the carboxy group of 6, which is one of the first nonpeptide antagonists reported by the Takeda group,12 upon the His6 imidazole ring and the C-terminal carboxy group of AII, respectively, and directed the benzyl group toward the N-terminus of AII. The SmithKline Beecham group19 suggested that the imidazole ring might not only mimic the peptide amide bond of AII but also act as a scaffold, locating the pharmacophores in favorable positions. They supposed that the butyl, carboxy, and 2-chlorobenzyl groups of 712 might correspond to the alkyl chain of Ile5, Cterminal carboxylic acid, and the side chain of Tyr4 in AII, respectively. Furthermore, the benzylcarboxylate and thienylmethyl acrylate moieties in 3 could overlay the side chain of Tyr4 and the C-terminal Phe8, respectively. On the other hand, the Glaxo group suggested that the carboxy group at the 5-position on the imidazole ring would not work as an ionic interaction with the AII receptor but as a hydrogen-bonding interaction.21

In a previous paper,22 we reported that imidazoles that had hydroxymethyl and carboxy groups at the 4and 5-positions, respectively, possessed potent antagonistic activity, which would be caused by hydrogen bonding or hydrophilicity of the hydroxymethyl group. Here we report on the synthesis of the imidazole antagonists possessing alkyl, alkenyl, and hydroxyalkyl substituents at the 4-position, their biological evaluation (SAR), and stereochemical comparison with the Cterminal pentapeptide of AII.

Yanagisawa et al.

Scheme 1a

a Reagents: (i) 6 N HCl; (ii) HCl/EtOH; (iii) R1MgX/Et O/ 2 CH2Cl2; (iv) POCl3/pyridine/benzene; (v) H2/Pd-C/MeOH; (vi) Ac2O; (vii) MeMgBr/THF; (viii) EtMgCl/THF/CH2Cl2; (ix) NaBH4/ EtOH.

Chemistry All the imidazole moieties referred to in this paper were derived from the imidazole-4,5-dicarbonitriles 8ac, which were easily prepared by heating ortho esters with diaminomaleonitrile, as shown in Scheme 1. Acid hydrolysis of 8a-c in 6 N HCl gave the dicarboxylic acids 9a-c. After esterification of 9a-c in ethanol in the presence of hydrogen chloride, the diesters 10a-c obtained were treated with 4 equiv of MeMgBr or EtMgCl, to afford the 4-(1-hydroxyalkyl)imidazoles 11a,ba,bb,c in 76-95% yields. Dehydration of 11ba with phosphoryl chloride and pyridine gave the 4-isopropenylimidazole 12, which was converted to the 4-isopropylimidazole 13 by hydrogenation on Pd-C. Decarboxylation23 of 9b was carried out by boiling in Ac2O, to give the monocarboxylic acid 14, which was esterified with HCl-ethanol to give 15. Grignard reaction of 8b with 3 equiv of MeMgBr afforded the 4-acetylimidazole 16. Acid hydrolysis of 16, followed by esterification, gave 18. Reaction of 18 with EtMgBr afforded 11bc. Reduction of 18 with NaBH4 provided the 4-(1-hydroxyethyl)imidazole 19. Alkylation of 10b with the biphenyl 20a13c furnished the (biphenylylmethyl)imidazole 21a, as shown in Scheme 2. Similarly, alkylation of 11a,ba,bb,bc,c and 13 with 20a afforded 21b-g, and alkylation of 10b, 11a,ba,c and 13 with 20b13c gave 22a-e. Removal of the tert-butyl group in 21a-g with 4 N HCl in dioxane, followed by alkaline hydrolysis, afforded the target dicarboxylic acids 25a-g. Detritylation of 22a-e with 25% aqueous AcOH, followed by alkaline hydrolysis, provided the desired compounds 26a-e.

Imidazolecarboxylic Acids as AII Receptor Antagonists

Scheme 2a

Journal of Medicinal Chemistry, 1996, Vol. 39, No. 1 325

Scheme 3a

a Reagents: (i) ButOK/AcNMe ; (ii) 4 N HCl/dioxane or 25% 2 aqueous AcOH; (iii) LiOH/dioxane/H2O.

a Reagents: (i) ButOK/AcNMe ; (ii) 21a-g f 23a-g, 4 N HCl/ 2 dioxane; 22a-e f 24a-e, 25% aqueous AcOH; (iii) LiOH/dioxane/ H2O.

Alkylation of the imidazole 15, bearing no substituent at the 5-position, with 20a yielded a mixture of the regioisomers 27a and 28a, in a ratio of 1:3, as shown in Scheme 3. Alkylation of 12 and 19, which have isopropenyl and 1-hydroxyethyl groups at the 4-position, respectively, afforded predominantly 27b,c with minor products 28b,c. Similar results were obtained on alkylation of 12, 15, and 19 with 20b: i.e., 15 afforded 29a and 30a in a ratio of 1:3, and 12 and 19 provided predominantly 29b,c with minor products 30b,c. Removal of the protecting groups of 27a-c, 28a, 29a-c, and 30a gave the target compounds 33a-c, 37, 34ac, and 38, respectively, by the same procedure described in Scheme 2. The structures of the alkylated imidazoles in Schemes 2 and 3 were determined by proton NMR spectra and X-ray analysis. The methylene protons of the biphenylylmethyl groups of the imidazole-5-carboxylates 21a-g, 22a-e, 27a-c, and 29a-c were observed at δ 5.3-5.7. On the other hand, the methylene protons of the imidazole-4-carboxylates 28a-c and 30a-c were at δ 5.0-5.3. The 0.3-0.5 ppm downfield shifts observed in the imidazole-5-carboxylates, in comparison with the imidazole-4-carboxylates, are caused by the anisotropic effect of the 5-ester group. The structure of 22c was assuredly confirmed by X-ray analysis of 26c, which was obtained from 22c as described above. The X-ray-determined structure is shown in Figure 1a. The X-ray analysis showed a protonation of the nitrogen atom at the 3-position on the imidazole ring, a carboxylate anion form of the carboxy group at the 5-position, and the presence of an intramolecular hydrogen bond between the hydroxy group at the 4-position and the 5-carboxylate anion.

Modifications22 of the ester groups on the imidazole rings are illustrated in Scheme 4. Reduction of 21a and 22a with diisobutylaluminum hydride (DIBAH) gave 4-(hydroxymethyl)imidazoles 39 and 40, respectively. On the other hand, reduction of 21a with lithium tritert-butoxyaluminohydride afforded predominantly 5-(hydroxymethyl)imidazole 45, with minor product 39, in a ratio of 6.6:1, and 22a provided 46 and 40 in a ratio of 9.5:1.24 The structure of 39 was determined by X-ray analysis; the result is shown in Figure 1b. The X-ray analysis showed the presence of an intermolecular hydrogen bond between the hydroxy group at the 4-position and the nitrogen atom at the 3-position. The structures of the other reduced products were deduced by proton NMR spectra, that is, the methylene protons of the biphenylylmethyl groups of 39 and 40 were observed at δ 5.61 and 5.47, respectively, whereas those of 45 and 46 were shown at higher fields, δ 5.26 and 5.08, respectively, as observed in the products of Schemes 2 and 3. Further reduction of 45 with DIBAH gave the diol 51. Removal of the protecting groups in 39, 40, 45, 46, and 51 afforded the objective compounds 43, 44, 49, 50, and 52, respectively. The 4-methyl- and -ethylimidazoles 57a,b and 58a,b were prepared by dehydroxylation of 27c, 29c, 39, and 40, as shown in Scheme 5. Esterification of 39 with ethyl chloroglyoxylate, followed by heating with tributyltin hydride, gave the dehydroxylated imidazole 53a, which was converted to 57a by removal of the protecting groups. The same procedure was done on 27c, 29c, and 40 to give the desired compounds 57b and 58a,b. Preparation of prodrugs 63a-d, 64a,b, and 68, which were expected to improve bioavailability, is illustrated in Scheme 6. Alkaline hydrolysis of 21c and 22c gave the imidazole-5-carboxylic acids 59 and 60, respectively. Esterification of 59 and 60 with appropriate alkyl halides, followed by removal of the tert-butyl or trityl group, gave 63a-d and 64a,b which are the prodrugs of the carboxy group at the 5-position on the imidazole ring. The X-ray-determined structure of 64a (CS-866), which is one of the most promising compounds, is shown

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Scheme 4a

a Reagents: (i) Bui AlH/toluene; (ii) 4 N HCl/dioxane or 25% 2 aqueous AcOH; (iii) LiOH/dioxane/H2O; (iv) LiAl(OBut)3H/THF.

Scheme 5a

Figure 1. X-ray-determined structures of 26c (a), 39 (b), and 64a (c).

in Figure 1c. The prodrug 68 of the carboxybiphenyl moiety was prepared as follows. Benzylation of 59, followed by removal of the tert-butyl group, gave 66. Pivaloyloxymethylation of 66 with (pivaloyloxy)methyl chloride, followed by removal of the benzyl group by hydrogenolysis, afforded 68. Biological Activities Binding Affinities to AII (AT1) Receptor (in Vitro Activities) and Antagonistic Activities by Intravenous Administration (in Vivo Activities). Compounds prepared in Schemes 2-5 were evaluated for in vitro and in vivo AII antagonistic activities. The in vitro activities were determined by a conventional ligand-binding assay on a bovine adrenal cortex using [125I]AII and are expressed as IC50 values. The in vivo

a Reagents: (i) ClCOCO Et/Et N/CH Cl then Bu SnH/toluene; 2 3 2 2 3 (ii) 4 N HCl/dioxane or 25% aqueous AcOH; (iii) NaOH/dioxane/ H2O.

activity was determined by the inhibition of the pressor response induced by infusion of AII in anesthetized normotensive rats. Compounds were administered intravenously, and inhibitory effects were estimated at 2 min after administration of the test compounds. The potency is expressed in terms of ID50 values. The resulting in vitro and in vivo activities are shown in Table 1, in which the activities are compared with those of 1 and 2 and their carboxybiphenyl analogues 69 and 70. Antagonistic Activities by Oral Administration. In order to evaluate for oral activities, 25c, 26c, 34c, and 44, which showed potent antagonistic activities by


Scheme 6a

a Reagents: (i) LiOH/dioxane/H O; (ii) RCl/K CO /AcNMe ; (iii) 2 2 3 2 4 N HCl/dioxane or 25% aqueous AcOH; (iv) PhCH2Br/K2CO3/ t AcNMe2; (v) Bu CO2CH2Cl/K2CO3/AcNMe2; (vi) H2/Pd-C/EtOH.

intravenous administration, and the prodrugs26 of 25c and 26c were orally administered to conscious normotensive rats. After administration of the test compounds, AII was administered repeatedly at appropriate intervals and the AII pressor responses were measured. The inhibitory effects, recorded at 1, 3, and 6 h after oral administrarion of the test compounds, are shown in Table 2 and are compared with those of 1 and 2. Molecular Modeling Conformational Analysis of 26c. Calculation on 26c was carried out on structure 71, in which the carboxy group and imidazole ring take the forms of a carboxylate anion and a protonated imidazole ring, respectively, as was found in the X-ray-determined structure (Figure 1a), and the tetrazole group is in an anion form in order to reduce the computational burden by introducing C2 symmetry into the ring. Geometry optimization of 71 was carried out using the PM3 method of the MOPAC program.27 First, the threedimensional structures of 72 and 73 were examined to find the stable substructure conformations of 71. The torsional angles of τ1-τ8 were incremented by 30°, 120°, 30°, 120°, 30°, 30°, 30°, and 30°, respectively, in the generation of initial conformations. After the geometry optimization, 43 and 2 distinct conformers with a relative energy (difference from the lowest) of ∆E < 7.0 kcal/mol were obtained for 72 and 73, respectively. As for the torsional angle of τ2 to locate the hydroxyl hydrogen, only the most preferable angles were taken into account. Initial conformations of 71 were then generated by combining the preferred torsional angles of τ1-τ8 obtained above. Geometry optimization of those

Journal of Medicinal Chemistry, 1996, Vol. 39, No. 1 327 Table 1. In Vitro and in Vivo AII Antagonist Properties of Imidazoles

compd

R1

R2

R3

Y

in vitro IC50a (nM)

33a 57a 57b 25e 33b 43 33c 25c 25f 25g 41 23c 37 25a 49 52 25b 25d 34a 58a 58b 26e 34b 44 34c 26c 24c 38 26a 50 26b 26d 1 2 69 70

Pr Pr Pr Pr Pr Pr Pr Pr Pr Pr Pr Pr Pr Pr Pr Pr Et Bu Pr Pr Pr Pr Pr Pr Pr Pr Pr Pr Pr Pr Et Bu Bu Bu Bu Bu

H Me Et CHMe2 C(dCH2)Me CH2OH CHMeOH CMe2OH CMeEtOH CEt2OH CH2OH CMe2OH CO2H CO2H CO2H CH2OH CMe2OH CMe2OH H Me Et CHMe2 C(dCH2)Me CH2OH CHMeOH CMe2OH CMe2OH CO2H CO2H CO2H CMe2OH CMe2OH Cl Cl Cl Cl

CO2H CO2H CO2H CO2H CO2H CO2H CO2H CO2H CO2H CO2H CO2Et CO2Et H CO2H CH2OH CH2OH CO2H CO2H CO2H CO2H CO2H CO2H CO2H CO2H CO2H CO2H CO2Et H CO2H CH2OH CO2H CO2H CH2OH CO2H CH2OH CO2H

CO2H CO2H CO2H CO2H CO2H CO2H CO2H CO2H CO2H CO2H CO2H CO2H CO2H CO2H CO2H CO2H CO2H CO2H Tzc Tz Tz Tz Tz Tz Tz Tz Tz Tz Tz Tz Tz Tz Tz Tz CO2H CO2H

60 110 100 360 270 58 28 28 51 110 840 600 45 000 320 20 000 2700 37 30 9.1 11 12 46 50 6.9 4.9 8.1 200 14 000 100 4600 5.3 9.5 120d 22d 490d 130d

in vivo ID50b (mg/kg) 0.22 0.096 0.27 0.14 0.37 0.069 0.062 0.056 0.074 0.11 1.5 1.2 8.2 0.67 8.8 2.1 0.30 0.066 0.018 0.028 0.019 0.014 0.012 0.0062 0.0063 0.0079 0.32 2.4 0.031 1.7 0.019 0.017 0.30 0.10 2.3 0.21

a IC 50 values were determined by a radioligand-binding assay using [125I]AII (0.1 nM) and bovine adrenal cortex. b ID50 values were determined by inhibitory activity to the AII-induced pressor response. Test compounds were intravenously administered. c Tetrazol-5-yl. d See ref 25.

structures gave 28 stable conformers with the ∆E < 7 kcal/mol. These conformers were classified into six groups, neglecting the conformations of the propyl substituent at the 2-position. The most stable conformations of each group are listed in Table 3. Conformational Analysis of Acetylated C-Terminal Pentapeptide of AII. Considering the computational ease, calculations were performed on the acetyl derivative of the C-terminal pentapeptide of AII, AcTyr-Ile-His-Pro-Phe-CO2- (74), which is considered to be the target sequence of nonpeptide antagonists as mentioned below. The two initial conformations of 74 were taken from TAA28 (Tyr145-Phe-His-Pro-Phe149) and AK329 (Trp127-Ile-His-Pro-Gly131) which were selected by the sequence homology search of Protein Data Bank30 at Brookhaven National Laboratory. After mutation of those two structures into 74, energy minimization was performed using the QUANTA/CHARMm program.31 The pentapeptide is yet so flexible with 17 rotatable

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Table 2. Inhibition of the AII-Induced Pressor Response by Imidazole Derivatives in Conscious, Normotensive Rats by Oral Administration

inhibition (%) compd

R2

R3

25c

CMe2OH

H

CO2H

23c

CMe2OH

Et

CO2H

63a 63b

CMe2OH CMe2OH

MDOa POMb

CO2H CO2H

63c 63d 68 44 34c 26c

CMe2OH CMe2OH CMe2OH CH2OH CHMeOH CMe2OH

IPMc PHTd H H H H

CO2H CO2H CO2POM Tze Tz Tz

24c 64a

CMe2OH CMe2OH

Et MDO

Tz Tz

64b

CMe2OH

POM

Tz

1

Cl

(CH2OH) Tz

2

Cl

H

Y

Tz

dose at at at (mg/kg) 1 h 3 h 6 h 1 3 10 3 10 1 0.3 1 3 1 1 1 0.3 0.3 0.3 1 1 0.03 0.1 0.3 0.03 0.1 0.3 1 3 10 1 3 10

50 78 88 26 40 72 43 52 85 71 73 20 76 80 83 98 70 32 66 78 29 75 76 12 38 69 74 81 89

42 67 70 28 70 70 32 61 85 71 77 35 75 78 77 98 72 39 77 88 41 84 85 12 35 77 72 81 93

12 48 56 25 75 70 35 75 87 71 74 40 65 69 78 93 76 55 83 92 43 70 90 32 65 85 70 81 89

a (5-Methyl-2-oxo-1,3-dioxol-4-yl)methyl. b (Pivaloyloxy)methyl. c [(Isopropoxycarbonyl)oxy]methyl. d Phthalidyl. e Tetrazol-5yl.

Table 3. Representative Stable Conformers of 71 with ∆E < 7.0 kcal/mol con∆E torsional angles (deg) former (kcal/ no. τ1 mol) τ2 τ3 τ4 τ5 τ6 τ7 τ8 1 2 3 4 5 6

138 58 80 180 53 -117 -89 160 -138 -56 80 180 53 63 84 19 -138 -58 -83 -179 -53 117 88 22 139 56 -83 -179 -51 118 90 18 13 159 82 180 54 62 84 21 11 159 -84 -179 -53 118 90 18

0.0 0.6 1.5 2.1 3.2 4.9

bonds, shown as thick lines, that the random sampling approach was employed using the “filter search” option of the QUANTA/CHARMm program. Starting from the two conformations derived from TAA and AK3, 100 stable conformations were obtained from each initial conformation. The conformational energies of those conformers ranged from -65.7 to -38.5 kcal/mol. Results and Discussion SAR: (i) In Vitro Activities. In the carboxybiphenyl series (Y ) CO2H), the imidazole-5-carboxylic acid 33a (IC50 ) 60 nM), which has no substituent at the 4-position, had a 2-fold stronger binding affinity than

1 and 70 but was less potent than 2. Introduction of alkyl or alkenyl groups at the 4-position lowered the binding affinity. The relative order of binding affinities to the receptor was H (33a) > Me (57a) ) Et (57b) > C(dCH2)Me (33b) > CHMe2 (25e), showing that a bulky alkyl or alkenyl group is unfavorable for binding to the receptor. Replacement of the alkyl group with a hydroxyalkyl group at the 4-position led to an improvement of the activity as seen in 43, 33c, and 25c. A higher improvement ratio was observed in bulkier R2 [Me (57a) vs CH2OH (43), 1.9-fold; Et (57b) vs CHMeOH (33c), 3.6-fold; CHMe2 (25e) vs CMe2OH (25c), 13-fold], which means that the hydroxy group plays an important role in the binding affinity and compensates the disadvantage of lipophilicity of the bulky alkyl group. The hydroxymethyl derivative 43, which is smaller than 33c, and compounds 25f,g, having bulky substituents at the 4-position compared with 25c, had weak binding affinities compared with 33c and 25c. These results show that a medium-sized hydroxyalkyl group, such as CHMeOH and CMe2OH, is favorable for the substituent at the 4-position. Esterification (23c, 41) of the carboxy groups at the 5-position of 25c and 43 markedly decreased the activity, indicating that an ionizable group is favorable for the binding affinity. Imidazole4-carboxylic acids 37, 25a, and 49 showed poor activities, which suggests that a polar group, such as CO2H, is unfavorable at the 4-position. Imidazole-4,5-dimethanol 52, which has a hydroxymethyl group at the 4-position instead of the chlorine atom of 69, had a weak binding affinity compared with 69. It is interesting that the hydroxyalkyl substituent at the 4-position with the carboxy group at the 5-position, not with the ester and hydroxymethyl group, contributes to potentiation of activity. This contribution may be brought about by the intramolecular hydrogen bond between the hydroxyalkyl group and the carboxy group as seen in the X-raydetermined structure of Figure 1a. Shortening (25b) or elongating (25d) the alkyl group at the 2-position of 25c resulted in a slight loss of antagonistic activities. The 2-butylimidazole 25d, which is the 4-(1-hydroxy1-methylethyl) analogue of 70, had a 4-fold greater potent binding affinity than 70. Replacement of the carboxybiphenyl group with a tetrazolylbiphenyl group (Y ) tetrazolyl group) resulted


in an improved antagonistic activity; i.e., imidazole-5carboxylic acid 34a showed a 6.6-fold more potent binding affinity than the corresponding biphenylylcarboxylic acid 33a [34a (IC50 ) 9.1 nM) vs 33a (IC50 ) 60 nM)]. Alkylation (58a,b, 26e) or alkenylation (34b) at the 4-position reduced the binding affinity. The bulky alkyl substituents seemed to be disadvantageous for activity as observed in the carboxybiphenyl series. Introduction of a hydroxy group on the 4-alkyl group enhanced the activity. The bulkier substituent brought on higher improvement ratio on the activity [Me (58a) vs CH2OH (44), 1.6-fold; Et (58b) vs CHMeOH (34c), 2.4-fold; CHMe2 (26e) vs CMe2OH (26c), 5.7-fold]. This tendency was consistent with that in the carboxybiphenyl series. The ethyl ester 24c showed only onetwenty-fifth the binding activity of 26c, and imidazole4-carboxylic acids 26a, 38, and 50 had poor activities as observed in the carboxybiphenyl series. Replacement of the propyl group at the 2-position of 26c with an ethyl group (26b) improved the binding affinity. The 2-butyl derivative 26d, which is a 4-(1-hydroxy-1-methylethyl) analogue of 2, was less potent than 26c but more potent than 2. Compound 26c did not show antagonism against the AT2 receptor prepared from the cerebrum membrane at a concentration of 0.1 mM, which implies that 26c is the AT1 selective antagonist. (ii) In Vivo Activities. SAR on in vivo activities is expected to be different from that on in vitro activities because of the effect of metabolism and pharmacokinetics of test compounds. In the carboxybiphenyl series, 33a, bearing no substituent at the 4-position, had the same activity as 70, which has a chlorine atom at the 4-position. Although alkylation (57a, 57b, 25e) of 33a reduced the in vitro activities, their in vivo activities were comparable with or more potent than that of 33a. The most active derivative 57a (R2 ) Me) suppressed the AII-induced pressor response by twice as much as 33a. These results are presumably caused by metabolic instability of 33a and a high affinity of the alkyl antagonists toward the target organ. Isopropenylation (33b) reduced the potency by almost one-half that of 33a. The hydroxyalkyl derivatives (43, 33c, 25c) had more potent activities than the corresponding alkyl compounds [Me (57a) vs CH2OH (43), 1.4-fold; Et (57b) vs CHMeOH (33c), 4.3-fold; CHMe2 (25e) vs CMe2OH (25c), 2.5-fold], which is in agreement with their potent in vitro activities. More bulky hydroxyalkyl substituents (25f,g) diminished the potencies as seen in the in vitro activities. The weak in vivo activities of ethyl esters 41 and 23c, 4-carboxy compounds 37, 25a, and 49, and the 4,5dihydroxymethyl derivative 52 were consistent with their weak in vitro activities. The 2-ethyl derivative 25b had only one-fifth potency of 25c, while the 2-butyl derivative 25d showed almost the same potency as 25c. Replacement of the carboxybiphenyl group with a tetrazolylbiphenyl group led to a drastic increase in the in vivo activity. Compound 34a, bearing no substituent at the 4-position, inhibited the AII pressor response by 1 order of magnitude and is, therefore, more potent than the corresponding biphenylcarboxylic acid 33a. Alkylation (58a,b, 26e) and isopropenylation (34b) at the 4-position of 34a kept the in vivo activities at the level


of 34a. Introduction of a hydroxy group (44, 34c, 26c) to the alkyl group of 58a,b and 26e led to an enhancement in the activities by a 1.8-4.5-fold range as observed in the carboxybiphenyl series. These compounds had the most potent antagonistic activities in the compounds prepared. Esterification (24c) markedly diminished the activity. Except for 26a, 4-carboxy derivatives 38 and 50 had weak activities as predicted from their in vitro activities. The ethyl (26b) and butyl (26d) derivatives at the 2-position were less potent than 26c. Oral Activities. One of the pharmacological requirements of an antihypertensive agent is that it must be orally effective. Sometimes, agents, which have potent in vitro activities and significantly reduce blood pressure by intravenous administration, do not show sufficient efficacy by oral administration because of their low bioavailability, liability to metabolism, and high clearance. Compound 25c, which was the most potent compound in the carboxybiphenyl series, showed maximal suppression within 1 h after oral administration. Its potency was more than that of 1 but less than that of 2. The ID50 values of 25c by iv and po administrations were 0.056 and 1 mg/kg, respectively. The large ID50 value by po administration would be caused by poor bioavailability of 25c. In order to improve the bioavailability, 25c was esterified and these esters were evaluated for their ability to suppress the AII pressor response. Compound 25c has two carboxy groups on the imidazole ring and the biphenyl moiety. At first, the esters of the imidazole carboxy group were examined. The ethyl ester 23c suppressed the AII-induced pressor response less potently than 25c. This result means that the ethyl ester is not converted to the parent carboxylic acid in vivo by the esterase. The (5-methyl2-oxo-1,3-dioxol-4-yl)methyl (MDO) (63a), (pivaloyloxy)methyl (POM) (63b), [(isopropoxycarbonyl)oxy]methyl (63c), and phthalidyl (63d) esters, whose ester residues are known to be useful as prodrug residues of the β-lactam antibiotics,32 showed potent and long-lasting suppression of the AII-induced pressor response. Their potencies were the same as that of 2 and 3-fold more potent than that of 25c. On the other hand, the POM ester 68 at the carboxybiphenyl moiety gave poor inhibitory activity. In the tetrazolylbiphenyl series, imidazole-5-carboxylic acids 44, 34c, and 26c, which showed strong binding affinities and potent suppressive activities on the AIIinduced pressor response by intravenous administration, showed potent and long-lasting suppression of the AII-induced pressor response orally. These compounds suppressed the AII-induced pressor response in a range of 76-83% at a dose of 0.3 mg/kg. The time courses for suppression of these compounds are shown in Figure 2. Of these three compounds, 26c showed the most prolonged effect, which may be caused by its metabolic stability due to the hindrance of the tertiary alcohol and by its large lipophilicity compared with 34c and 44. Hereupon, the most favorable acid (26c) was esterified in order to improve bioavailability. Though the ethyl ester 24c was less active than the parent compound 26c, the MDO (64a) (CS-866) and POM (64b) esters had the most potent and durable inhibitory activities among the compounds tested by oral administration. Their poten-

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Figure 2. Time courses of 26c (O), 34c (b), and 44 (9) for inhibition of the AII (50 ng/kg, iv)-induced pressor response in conscious normotensive rats (n ) 3-5). The compounds were administered orally at a dose of 0.3 mg/kg.

Figure 3. Time courses of 1 (3 mg/kg, po, 9), 26c (0.3 mg/kg, po, O), and 64a (0.1 mg/kg, po, b) for inhibition of the AII (50 ng/kg, iv)-induced pressor response in conscious normotensive rats (n ) 3-5).

cies at 0.1 mg/kg dose were comparable to that at 0.3, 3, and 10 mg/kg doses of 26c, 2, and 1, respectively. The time course for inhibition of 64a (0.1 mg/kg, po) in comparison with 1 (3 mg/kg, po) and 26c (0.3 mg/kg, po) is shown in Figure 3. Superposition of 26c upon Acetylated C-Terminal Pentapeptide of AII. For the purpose of understanding the role of the 1-hydroxy-1-methylethyl group at the 4-position of 26c from the standpoint of stereochemistry, we studied stereochemical comparison of 26c with AII. The conformation bound to the receptor has not been determined definitely on account of its gross flexibility.33 In the early overlay studies17a,19 of the nonpeptide antagonists 6, 7, and 3 with AII, these antagonists overlapped with the C-terminal pentapeptide of AII, Tyr4-Ile5-His6-Pro7-Phe8. Garcia et al. reported the three-dimensional structure of the AII-Fab complex, in which the central AII sequence, Tyr4-Ile5-His6-Pro7, located in the deepest region of the combining site, and the terminal Phe8 were important residues for binding to the receptor.34 Consequently, studying the conformation of the C-terminal pentapeptide of AII, instead of AII, can be considered a logical approach to the overlay study. In practice, the acetyl derivative 74 of the C-terminal pentapeptide of AII was adopted for a molecular modeling study, in order to simplify the calculations.

Yanagisawa et al.

Each conformation of 74, obtained by the molecular modeling study, was examined graphically to determine whether it could be superposed on any of the six conformers of 71 listed in Table 3, with the tetrazole ring of 71 overlapped on either the C-terminal carboxy group or the phenolic hydroxy group of Tyr in 74. These acidic moities are supposed to be one of the most important pharmacophores. Several pairs that appeared to be well-superposed upon each other were selected and then subjected to a more unbiased superposition to maximize the overlap of the van der Waals volume of hydrophobic groups, i.e., carbon atoms and hydrogen atoms attached to them. Two kinds of superposed structures, A and B, with the most reasonable correspondence of functional groups are shown in Figure 4. Conformers 4 and 3 of 71 in Table 3 were employed in superpositions A and B, respectively. In the case of superposition A, the acidic moieties, tetrazole and carboxy groups, of 71 correspond to the phenolic hydroxy group of Tyr and the C-terminal carboxy group, respectively, and the hydrophobic moieties, the propyl, isopropyl, and terminal phenyl groups, agree with the alkylene chain of Pro, the phenyl group of Phe, and the phenyl group of Tyr, respectively. The imidazole ring of 71 has no directly corresponding group in 74 but seems to serve as a junction of the pharmacophore groups. In contrast with superposition A, the tetrazole and carboxy groups of superposition B correspond to the C-terminal carboxy group and the carbonyl group of Ile in 74, respectively. The propyl, isopropyl, terminal phenyl, and internal benzyl groups can be overlayed on the alkylene chain of Pro, the alkyl group of Ile, the phenyl group of Phe, and the imidazolylmethyl group of His, respectively. The nitrogen atom at the 3-position on the imidazole ring, which would interact with the AII receptor through hydrogen bonding,18 is located near the carbonyl oxygen of His. It is interesting in the superposition B that the 1-hydroxy-1-methylethyl group of 71 is positioned where the alkyl side chain and the peptide backbone carbonyl group of Ile are located. The hydrogen bond between the hydroxy group in the 4-substituent and the neighboring carboxy group in 71 seems to change the nature of the carboxy group to that of the corresponding carbonyl group of Ile from the electostatic point of view and to direct the isopropyl group toward the position occupied by the side chain of Ile. These speculations might account for the potent activities of 26c and the compounds having a hydroxyalkyl group at the 4-position. In superposition B we suppose that 71 would bind to the AII receptor through the following interactions: (i) hydrophobic bindings of the propyl chain at the 2-position, the isopropyl group at the 4-position, the terminal phenyl group of the substituent at the 1-position, and the benzyl group at the 1-position, which correspond to the alkylene group of Pro, the alkyl chain of Ile, the phenyl group of Phe, and the imidazolylmethyl group of His in AII, respectively, with lipophilic pockets of the receptor, (ii) an ionic bonding between the tetrazole group, corresponding to the C-terminal carboxy group of AII, and a cationic group of the receptor, and (iii) a hydrogen bonding between the carboxy group at the 5-position, corresponding to the carbonyl group of Ile, and a proton donor of the receptor. Hydrophobic



Figure 4. Stereoviews of superposition structures A and B. Thick lines indicate 71, and thin lines indicate 74. (a) Superposition A: The overlapped van der Waals volume of hydrophobic groups is 172 Å3. (b) Superposition B: The overlapped van der Waals volume of hydrophobic groups is 186 Å3.

interaction between the substituent at the 4-position and a postulated secondary lipophilic pocket in the AII receptor was proposed by the Searle group.35 A few groups17d,36 suggested that a character of the substituent at the 5-position as a hydrogen-bonding acceptor is favorable for binding to the receptor. These proposed interactions are well-consistent with our results. Furthermore, the conformation of 71 in superposition B is in fair agreement with that of a potent, conformationally restricted imidazole antagonist36b which would be the active conformation for interaction with the receptor. Conclusion 2-Alkyl-1-[[2′-carboxy(or tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole-5-carboxylic acids bearing alkyl, alkenyl, and hydroxyalkyl substituents at the 4-position have been synthesized and evaluated for in vitro and in vivo antagonisic activities on the AII receptor. In the carboxybiphenyl series, the most active compound, 25c, has propyl, 1-hydroxy-1-methylethyl, and carboxy groups at the 2-, 4-, and 5-positions, respectively. The low oral bioavailability of 25c was improved by esterification of the 5-carboxy group with MDO, POM, [(isopropoxycarbonyl)oxy]methyl, and phthalidyl groups. Replacement of the carboxybiphenyl group with a tetrazolylbiphenyl group enhanced the antagonistic activity. The most potent imidazoles were hydroxymethyl (44), 1-hydroxyethyl (34c), and (1-hydroxy-1-methyl)ethyl (26c) derivatives, which had 2.7-4.5 times stronger binding affinity than 2 and 15-24 times stronger affinity than 1, and they inhibited the AII-induced pressor response 13-16 times more potently than 2 by intravenous administration. Among them, 26c showed the most lasting antagonistic activity, when administered orally. The MDO (64a) (CS-866) and POM (64b) esters of 26c were the most promising antagonists prepared in the present study. These compounds suppressed the AII-induced pressor response 3 times more potently than 26c and 100 times more potently than 1 by oral administration. Stereochemical comparison of 26c with the acetylated C-terminal pentapeptide 74 of AII gave two reasonable superposition structures, A and B. Superposition B suggested that the substituent suitable for the 4-position would be a medium-sized hydrophobic group having a

hydroxy group which forms a hydrogen bond with the carboxy group at the 5-position. This hydrogen bonding would change the nature of the carboxy group at the 5-position to that of the carbonyl group of Ile and direct the isopropyl group toward the alkyl chain of Ile. The conformational analysis of the pentapeptide is incomplete in the following views: (i) the pentapeptide in AII was utilized in the computational modeling, (ii) the conformations of the pentapeptide sequences of TAA and AK3 were utilized in the conformational study of the pentapeptide of AII, and (iii) no solvents were taken into account in all the calculations. Despite such limitations, however, we are confident that our results would serve as a future working model for synthetic study of the next-generation antagonists. Experimental Section Melting points were determined on a Yanagimoto micromelting point apparatus and are uncorrected. Recrystallization solvents for analytical samples are described in parentheses after melting points. Proton NMR spectra were obtained on a JEOL EX270 instrument and are reported as δ values relative to Me4Si as the internal standard. The following abbreviations are used to describe peak patterns whenever appropriate: br ) broad, s ) singlet, d ) doublet, t ) triplet, q ) quartet, qi ) quintet, sx ) sextet, sp ) septet, m ) multiplet. IR spectra were taken on a JASCO FT/IR8900 spectrometer. The abbreviations sh and wk indicate shoulder and weak, respectively. Elemental analyses were performed by the Institute of Science and Technology, Inc., and the results obtained are within (0.4% of the theoretical values unless indicated otherwise. Flash column chromatography was done on Merck silica gel 60 (230-400 mesh). Isopropyl ether, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, and dimethyl sulfoxide are abbreviated as IPE, THF, DMF, DMA, and DMSO, respectively. 2-Propylimidazole-4,5-dicarbonitrile (8b).37 A mixture of diaminomaleonitrile (24.1 g, 0.162 mol) and trimethyl orthobutyrate (15.95 g, 0.148 mol) in CH3CN (95 mL) was stirred under reflux for 5 h. After the solvent was evaporated in vacuo, the residue was diluted with xylene (87 mL) and the solution was refluxed for 7 h. After cooling in an ice bath, the precipitates were collected by filtration: yield 18.7 g (96%); mp 141-144 °C. Similarly, 8a,c were prepared using trimethyl orthopropionate and trimethyl orthovalerate, respectively, instead of trimethyl orthobutyrate. 8a: 97%; mp 179-181 °C.38 8c: 79%; mp 111-112 °C. Anal. (C9H10N4) C,H,N.

332


2-Propylimidazole-4,5-dicarboxylic Acid (9b).39 A solution of 8b (39.5 g, 0.247 mol) in 6 N HCl (360 mL) was refluxed for 8 h. After cooling, the precipitates were collected by filtration and washed with water and acetone: yield 39.3 g (80%); mp 261-263 °C. Similarly, 9a,c were prepared. 9a: 55%; mp 265-268 °C.40 9c: 69%; mp 261-263 °C.40 Diethyl 2-Propylimidazole-4,5-dicarboxylate (10b). To a suspension of 9b (120 g, 0.606 mol) in EtOH (1.3 L) was bubbled HCl gas at room temperature for 3 h. The resulting solution was allowed to stand overnight at room temperature and concentrated in vacuo. The residue was dissolved in EtOAc and aqueous NaHCO3 and neutralized with powdered NaHCO3. The organic phase was separated, dried over MgSO4, and concentrated in vacuo to give crystalline 10b, which was washed with IPE-hexane: yield 147.8 g (86%); mp 84-86 °C (EtOAc-IPE); NMR (CDCl3) 0.97 (3H, t, J ) 7.5 Hz), 1.38 (6H, t, J ) 7 Hz), 1.79 (2H, sx, J ) 7.5 Hz), 2.78 (2H, t, J ) 7.5 Hz), 4.39 (4H, q, J ) 7 Hz); IR (KBr) 2609, 1749, 1724 (sh) cm-1. Anal. (C12H18N2O4) C,H,N. Similarly, 9a,c and 14 were esterified to afford 10a,c and 15, respectively. 10a: 85%; mp 88-90 °C (EtOAc-IPE).38 10c: 85%; mp 83-84 °C. Anal. (C13H20N2O4) C,H,N. 15: 56%; mp 108-110 °C. Anal. (C9H14N2O2) C,H,N. Ethyl 4-(1-Hydroxy-1-methylethyl)-2-propylimidazole5-carboxylate (11ba). To a stirred solution of 3 M MeMgBr in Et2O (250 mL, 0.75 mol) was added a solution of 10b (45.7 g, 0.18 mol) in CH2Cl2 (120 mL) at 0-10 °C under N2. The mixture was stirred at 10-15 °C for 1 h and then diluted with EtOAc (0.5 L) and aqueous NH4Cl (0.3 L), successively. The organic phase was separated, washed with brine, dried over MgSO4, and concentrated in vacuo to give a syrup, which was crystallized in IPE-hexane: yield 40.9 g (95%); mp 101-102 °C (IPE); NMR (CDCl3) 0.97 (3H, t, J ) 7.5 Hz), 1.36 (3H, t, J ) 7 Hz), 1.62 (6H, s), 1.74 (2H, sx, J ) 8 Hz), 2.68 (2H, t, J ) 8 Hz), 4.36 (2H, q, J ) 7 Hz); IR (KBr) 3373, 1718 (sh), 1676 cm-1. Anal. (C12H20N2O3) C,H,N. Similarly, 11a,bb,c were prepared. 11a: 76%; mp 191192 °C (EtOAc). Anal. (C11H18N2O3) C,H,N. 11bb: 83%; syrup. Anal. (C14H24N2O3‚1/2H2O) C,H,N. 11c: 78%; syrup. Anal. (C13H22N2O3) C,H,N. Ethyl 4-Isopropenyl-2-propylimidazole-5-carboxylate (12). To a solution of 11ba (10.0 g, 41.6 mmol) in benzene (200 mL) was dropwise added POCl3 (8.53 mL, 91.5 mmol), and the mixture was refluxed for 1 h. Pyridine (10.0 mL, 124 mmol) was added to the reaction mixture at room temperature, and the mixture was refluxed for 1 h. After removal of the solvent by evaporation in vacuo, the residue was dissolved in EtOAc and aqueous NaHCO3 and neutralized with powdered NaHCO3. The organic phase was separated, washed with aqueous NaCl, dried over Na2SO4, and concentrated in vacuo to give a crystalline residue, which was washed with IPE: yield 8.39 g (91%); mp 131-131.5 °C (EtOAc); NMR (CDCl3) 0.99 (3H, t, J ) 7.5 Hz), 1.36 (3H, t, J ) 7.5 Hz), 1.77 (2H, sx, J ) 7.5 Hz), 2.19 (3H, s), 2.70 (2H, t, J ) 7.5 Hz), 4.35 (2H, q, J ) 7.5 Hz), 5.31-5.32 (2H, m), 5.55 (1H, br s); IR (KBr) 1714 cm-1. Anal. (C12H18N2O2) C,H,N. Ethyl 4-Isopropyl-2-propylimidazole-5-carboxylate (13). A mixture of 12 (5.39 g, 24.2 mmol) and 10% Pd-C (0.54 g) in MeOH (50 mL) was stirred at room temperature under a hydrogen atmosphere for 3 h. The catalyst was filtered off, and the filtrate was concentrated in vacuo to give crystalline 13, which was washed with IPE: yield 5.04 g (93%); mp 133136 °C (EtOAc-hexane); NMR (CDCl3) 0.96 (3H, t, J ) 7.5 Hz), 1.29 (6H, d, J ) 7 Hz), 1.34 (3H, t, J ) 7 Hz), 1.75 (2H, sx, J ) 7.5 Hz), 2.72 (2H, t, J ) 7.5 Hz), 3.66 (1H, sp, J ) 7 Hz), 4.32 (2H, q, J ) 7 Hz); IR (KBr) 1708, 1699 cm-1. Anal. (C12H20N2O2) C,H,N. 2-Propylimidazole-4-carboxylic Acid (14). A suspension of 9b (24.0 g, 0.121 mol) in Ac2O (700 mL) was stirred under reflux for 33 h. After removal of the solvent by evaporation in vacuo, the residue was mixed with H2O (250 mL), and the mixture was refluxed for 3 h. The solvent was evaporated to give a crystalline residue, which was washed with EtOHEt2O: yield 14.5 g (78%); mp 242-244 °C dec (EtOH); NMR (DMSO-d6) 0.88 (3H, t, J ) 7.5 Hz), 1.58-1.74 (2H, m), 2.59

Yanagisawa et al. (2H, t, J ) 7.5 Hz), 7.54 (1H, s), 9.70-12.00 (1H, br s); IR (KBr) 1638 cm-1. Anal. (C7H10N2O2) C,H,N. 4-Acetyl-2-propylimidazole-5-carbonitrile (16). To a solution of 8b (10.0 g, 64.2 mmol) in THF (100 mL) was dropwise added a solution of 1 M MeMgBr in THF (194 mL, 194 mmol) at 10-15 °C under N2. After stirring at 10-15 °C for 0.5 h, the reaction mixture was diluted with EtOAc (200 mL) and saturated aqueous NH4Cl (200 mL), successively, and then acidified with aqueous KHSO4. The organic phase was separated, dried over MgSO4, and concentrated in vacuo. The residue was purified by flash column chromatography (EtOAc/ hexane, 1:1) to give crystalline 16: yield 9.18 g (81%); mp 9394 °C (IPE-hexane); NMR (CDCl3) 1.00 (3H, t, J ) 7.5 Hz), 1.81 (2H, sx, J ) 7.5 Hz), 2.72 (3H, s), 2.79 (2H, t, J ) 7.5 Hz), 10.70 (1H, br s); IR (KBr) 3430, 2238, 1680 cm-1. Anal. (C9H11N3O) C,H,N. Ethyl 4-Acetyl-2-propylimidazole-5-carboxylate (18). A solution of 16 (4.00 g, 23.2 mmol) in 6 N HCl (60 mL) was refluxed for 8 h. After evaporation of the solvent in vacuo followed by removal of a trace of H2O by codistillation with EtOH, the residual solid 17 was dissolved in EtOH (60 mL). To the solution was bubbled HCl gas at room temperature for 20 min. After standing at room temperature for 16 h, the solution was concentrated in vacuo. The residue was dissolved in EtOAc and aqueous NaHCO3 and neutralized with powdered NaHCO3. The organic phase was separated, dried over MgSO4, and concentrated in vacuo. The residue was purified by flash column chromatography (EtOAc/hexane, 1:1) to give crystalline 18: yield 3.07 g (59%); mp 80.5-82 °C (IPEhexane); NMR (CDCl3) 0.98 (3H, t, J ) 7.5 Hz), 1.43 (3H, t, J ) 7 Hz), 1.79 (2H, sx, J ) 7.5 Hz), 2.76 (3H, s), 2.77 (2H, t, J ) 7.5 Hz), 4.45 (2H, q, J ) 7 Hz), 10.44 (1H, br s); IR (KBr) 3426, 1725, 1665 cm-1. Anal. (C11H16N2O3) C,H,N. Ethyl 4-(1-Hydroxy-1-methylpropyl)-2-propylimidazole5-carboxylate (11bc). To a solution of 18 (460 mg, 2.05 mmol) in THF (5 mL) was added a solution of 2 M EtMgCl in THF (2.25 mL, 4.5 mmol) at -45 to -40 °C under N2. After stirring at -40 °C for 2.5 h, EtOAc and saturated aqueous NH4Cl were added, and then the mixture was stirred for 0.5 h. The organic phase was separated, dried over Na2SO4, and concentrated in vacuo. The residue was purified by flash column chromatography (MeOH/CH2Cl2, 1:30) to give 11bc as a syrup: yield 357 mg (69%); NMR (CDCl3) 0.86 (3H, t, J ) 7.5 Hz), 0.92 (3H, t, J ) 7.5 Hz), 1.28 (3H, t, J ) 7.5 Hz), 1.59 (3H, s), 1.72 (2H, sx, J ) 7.5 Hz), 1.77-1.90 (1H, m), 1.902.05 (1H, m), 2.66 (2H, t, J ) 7.5 Hz), 4.31 (2H, q, J ) 7.5 Hz), 6.06 (1H, br s); IR (liquid film) 3196, 1711 (sh), 1676 cm-1. Anal. (C13H22N2O3) H,N; C: calcd, 61.39; found, 60.88. Ethyl 4-(1-Hydroxyethyl)-2-propylimidazole-5-carboxylate (19). To a solution of 18 (1.50 g, 6.69 mmol) in EtOH (15 mL) was added NaBH4 (125 mg, 3.30 mmol), and the mixture was stirred at room temperature for 0.5 h. After adding acetone (2 mL), followed by stirring for 10 min, the reaction solution was concentrated in vacuo. The residue was purified by flash column chromatography (MeOH/CH2Cl2, 1:10) to give crystalline 19: yield 1.32 g (87%); mp 149-150.5 °C (EtOAc-IPE); NMR (CDCl3) 0.97 (3H, t, J ) 7.5 Hz), 1.37 (3H, t, J ) 7.5 Hz), 1.53 (3H, d, J ) 6.5 Hz), 1.75 (2H, sx, J ) 7.5 Hz), 2.69 (2H, t, J ) 7.5 Hz), 4.36 (2H, q, J ) 7.5 Hz), 5.23 (1H, q, J ) 6.5 Hz); IR (KBr) 3101, 1706 cm-1. Anal. (C11H18N2O3) C,H,N. Diethyl 1-[[2′-(tert-Butoxycarbonyl)biphenyl-4-yl]methyl]-2-propylimidazole-4,5-dicarboxylate (21a). After adding KOBut (561 mg, 5 mmol) to a solution of 10b (1.27 g, 5 mmol) in DMA (13 mL) under ice cooling, followed by stirring for 10 min, a solution of tert-butyl 4′-(bromomethyl)biphenyl2-carboxylate (20a) (2.08 g, 6 mmol) in DMA (20 mL) was added, and the mixture was stirred at room temperature for 1 h. EtOAc and H2O were added. The organic phase was separated, dried over MgSO4, and concentrated in vacuo. The residue was purified by flash column chromatography (EtOAc/ hexane, 1:2) to give 21a: yield 2.61 g (quantitative); mp 6567 °C (IPE-hexane); NMR (CDCl3) 0.96 (3H, t, J ) 8 Hz), 1.26 (9H, s), 1.27 (3H, t, J ) 7.5 Hz), 1.39 (3H, t, J ) 7.5 Hz), 1.76 (2H, sx, J ) 8 Hz), 2.67 (2H, t, J ) 8 Hz), 4.27 (2H, q, J ) 7.5 Hz), 4.39 (2H, q, J ) 7.5 Hz), 5.47 (2H, s), 7.03 (2H, d, J ) 8

Imidazolecarboxylic Acids as AII Receptor Antagonists Hz), 7.25-7.73 (3H, m), 7.42 (1H, t, J ) 7.5 Hz), 7.47 (1H, t, J ) 7.5 Hz), 7.77 (1H, d, J ) 7.5 Hz); IR (KBr) 1709 cm-1. Anal. (C30H36N2O6) C,H,N. Similarly, 21b-g and 22a-e were prepared. 21b: 80%; mp 88-89 °C (IPE). Anal. (C29H36N2O5) C,H,N. 21c: 85%; mp 91 °C (hexane). Anal. (C30H38N2O5) C,H,N. 21d: 71%; mp 94-96 °C (hexane). Anal. (C31H40N2O5) C,H,N. 21e: syrup; quantitative. Anal. (C30H38N2O4) C,H,N. 21f: syrup; 97%. Anal. (C31H40N2O5) C,H,N. 21g: syrup; 71%. Anal. (C32H42N2O5) C,N; H: calcd, 7.92; found, 7.37. 22a: 89%; mp 144145 °C (EtOAc). Anal. (C45H42N6O4) C,H,N. 22b: 81%; mp 152-153 °C dec (EtOAc). Anal. (C44H42N6O3) C,H,N. 22c: 80%; mp 165-166 °C dec (IPE-hexane). Anal. (C45H44N6O3) C,H,N. 22d: 88%; mp 118-121 °C dec (IPE). Anal. (C46H46N6O3) C,H,N. 22e: 69%; mp 122-124 °C (IPE-hexane). Anal. (C45H44N6O2) C,H,N. Ethyl 1-[[2′-(tert-Butoxycarbonyl)biphenyl-4-yl]methyl]2-propylimidazole-5-carboxlate (27a) and Ethyl 1-[[2′(tert-Butoxycarbonyl)biphenyl-4-yl]methyl]-2-propylimidazole-4-carboxlate (28a). By the same procedure as described in the preparation of 21a, 15 (1.00 g, 5.49 mmol) was alkylated with 20a (2.29 g, 6.59 mmol). The products were subjected to flash column chromatography (EtOAc/hexane, 1:1) to give the following fractions. Faster moving fraction 27a: syrup; yield 602 mg (24%); NMR (CDCl3) 0.98 (3H, t, J ) 7.5 Hz), 1.22 (9H, s), 1.32 (3H, t, J ) 7.5 Hz), 1.72-1.88 (2H, m), 2.66 (2H, t, J ) 7.5 Hz), 4.25 (2H, q, J ) 7.5 Hz), 5.63 (2H, s), 7.01 (2H, d, J ) 8.5 Hz), 7.23-7.30 (3H, m), 7.34-7.51 (2H, m), 7.77 (1H, d,d, J ) 1.5, 7.5 Hz), 7.78 (1H, s); IR (liquid film) 1710 cm-1. Anal. (C27H32N2O4) C,H,N. Slower moving fraction 28a: syrup; yield 1.83 g (74%); NMR (CDCl3) 0.98 (3H, t, J ) 7.5 Hz), 1.27 (9H, s), 1.37 (3H, t, J ) 7.5 Hz), 1.70-1.90 (2H, m), 2.69 (2H, t, J ) 7.5 Hz), 4.36 (2H, q, J ) 7.5 Hz), 5.14 (2H, s), 7.12 (2H, d, J ) 8.5 Hz), 7.267.53 (5H, m), 7.54 (1H, s), 7.79 (1H, d,d, J ) 1.5, 7.5 Hz); IR (liquid film) 1709 cm-1. Anal. (C27H32N2O4) C,H,N. Similarly, 27b,c, 28b,c, 29a-c, and 30a-c were prepared. 27b: syrup; 72%. Anal. (C30H36N2O4) C,H,N. 28b: gum; 18%. Anal. (C30H36N2O4‚0.7H2O) C,H,N. 27c: 87%; syrup. Anal. (C29H36N2O5) C,H,N. 28c: 6%; mp 168-169 °C (IPE). Anal. (C29H36N2O5) C,H,N. 29a: 22%; mp 169-171 °C dec (IPE). Anal. (C42H38N6O2) C,H,N. 30a: 67%; mp 187-189 °C dec (EtOAc). Anal. (C42H38N6O2) C,H,N. 29b: 63%; amorphous solid. Anal. (C45H42N6O2) C,H,N. 30b: 29%; amorphous solid. Anal. (C45H42N6O2) C,H,N. 29c: 81%; mp 135-137 °C (EtOAc-IPE). Anal. (C44H42N6O3) C,H,N. 30c: 5%; mp 195196.5 °C (IPE). Anal. (C44H42N6O3‚1/2H2O) C,H,N. Diethyl 1-[(2′-Carboxybiphenyl-4-yl)methyl]-2-propylimidazole-4,5-dicarboxylate (23a). A solution of 21a (2.04 g, 3.92 mmol) in 4 N HCl in dioxane (10 mL) was allowed to stand at room temperature for 16 h. The solvent was removed by evaporation in vacuo to give the HCl salt of 23a as an amorphous powder: yield 1.96 g (quantitative). The HCl salt (0.30 g, 0.60 mmol) was dissolved in H2O (10 mL) and EtOAc (10 mL). To the solution was added NaHCO3 (0.27 g, 3.2 mmol), and then the mixture was adjusted to pH 3.0 with 1 N HCl. The organic phase was separated, dried over MgSO4, and concentrated in vacuo to give 23a: yield 0.27 g (96%); mp 166-169 °C (EtOAc-IPE); NMR (DMSO-d6) 0.88 (3H, t, J ) 7.5 Hz), 1.16 (3H, t, J ) 7 Hz), 1.27 (3H, t, J ) 7 Hz), 1.63 (2H, sx, J ) 7.5 Hz), 2.65 (2H, t, J ) 7.5 Hz), 4.19 (2H, q, J ) 7 Hz), 4.24 (2H, q, J ) 7 Hz), 5.46 (2H, s), 7.07 (2H, d, J ) 8 Hz), 7.31 (2H, d, J ) 8 Hz), 7.34 (1H, d,d, J ) 1, 8 Hz), 7.45 (1H, d,t, J ) 1, 8 Hz), 7.56 (1H, d,t, J ) 1, 8 Hz), 7.72 (1H, d,d, J ) 1, 8 Hz); IR (KBr) 3429, 2616, 2498, 1729, 1697 cm-1. Anal. (C26H28N2O6) C,H,N. Similarly, 23b-g, 31a-c, and 35 were prepared from the corresponding tert-butyl esters. 23b: 96%; mp 206-207 °C. Anal. (C25H28N2O5) C,H,N. 23c: 92%; mp 167-168 °C (EtOAc-IPE). Anal. (C26H30N2O5) C,H,N. 23d: 99%; mp 148-150 °C (EtOAc-IPE). Anal. (C27H32N2O5) C,H,N. 23e: 82%; mp 178-179 °C. Anal. (C26H30N2O4) C,H,N. 23f: 78%; mp 158-160 °C (EtOAc). Anal. (C27H32N2O5) C,H,N. 23g: 92%; mp 165-167 °C (EtOAc-IPE). Anal. (C28H34N2O5) C,H,N. 31a: 94%; mp 200-204 °C (EtOH-Et2O). Anal. (C23H24N2O4) C,H,N. 31b: 92%; mp 181-182 °C (EtOAc).

Journal of Medicinal Chemistry, 1996, Vol. 39, No. 1 333 Anal. (C26H28N2O4) C,H,N. 31c: obtained as HCl salt; 94%; mp 166-168 °C (EtOAc-IPE). Anal. (C25H29ClN2O5) C,H, Cl,N. 35: 90%; mp 220-224 °C (EtOH). Anal. (C23H24N2O4) C,H,N. Diethyl 2-Propyl-1-[(2′-1H-tetrazol-5-ylbiphenyl-4-yl)methyl]imidazole-4,5-dicarboxylate (24a). A solution of 22a (1.01 g, 1.38 mmol) in 25% aqueous AcOH (20 mL) was stirred at 80 °C for 2.5 h. To the reaction solution was added H2O (15 mL), and the mixture was cooled in an ice bath. The precipitates were filtered off and washed with 50% aqueous AcOH. The filtrate was concentrated to give the amorphous powder 24a, which was washed with IPE: yield 492 mg (72%); NMR (CDCl3) 0.89 (3H, t, J ) 7.5 Hz), 1.24 (3H, t, J ) 7.5 Hz), 1.29 (3H, t, J ) 7.5 Hz), 1.64 (2H, sx, J ) 7.5 Hz), 2.62 (2H, t, J ) 7.5 Hz), 4.25 (2H, q, J ) 7.5 Hz), 4.26 (2H, q, J ) 7.5 Hz), 5.40 (2H, s), 6.94 (2H, d, J ) 8 Hz), 7.13 (2H, d, J ) 8 Hz), 7.39 (1H, d, J ) 7 Hz), 7.52 (1H, t, J ) 7.5 Hz), 7.58 (1H, d,t, J ) 1, 7.5 Hz), 7.93 (1H, d,d, J ) 1, 7.5 Hz); IR (KBr) 3428, 1717 cm-1. Anal. (C26H28N6O4) C,H; N: calcd, 17.20; found, 16.78. Similarly, 24b-e, 32a-c, and 36 were prepared from the corresponding trityl derivatives. 24b: 95%; mp 170-172 °C dec (EtOAc). Anal. (C25H28N6O3) C,H,N. 24c: 92%; mp 162164 °C (EtOAc). Anal. (C26H30N6O3) C,H,N. 24d: 77%; mp 140-142 °C (EtOAc-IPE). Anal. (C27H32N6O3) C,H,N. 24e: 93%; 119-122 °C (EtOAc). Anal. (C26H30N6O2) C,H,N. 32a: 84%; mp 173-175 °C (EtOAc). Anal. (C23H24N6O2) C,H,N. 32b: 94%; mp 180-181.5 °C (EtOAc). Anal. (C26H28N6O2) C,H,N. 32c: 92%; mp 151-153 °C (EtOAc-Et2O). Anal. (C25H28N6O3) C,H,N. 36: 86%; mp 120-123 °C (EtOAc). Anal. (C23H24N6O2) C,H,N. 1-[(2′-Carboxybiphenyl-4-yl)methyl]-2-propylimidazole4,5-dicarboxylic Acid (25a). After stirring a suspension of 23a (1.50 g, 2.72 mmol) and LiOH‚H2O (684 mg, 16.3 mmol) in dioxane (15 mL) and H2O (15 mL) at room temperature for 5 h, dioxane was removed by distillation in vacuo. To the residual solution was added 1 N HCl (16.3 mL) to give precipitates which were collected by filtration: yield 1.11 g (quantitative); mp 201-202 °C (EtOH-EtOAc); NMR (DMSOd6) 0.81 (3H, t, J ) 7.5 Hz), 1.48 (2H, sx, J ) 7.5 Hz), 2.90 (2H, t, J ) 7.5 Hz), 6.00 (2H, s), 7.20 (2H, d, J ) 8 Hz), 7.297.36 (3H, m), 7.45 (1H, t, J ) 8 Hz), 7.57 (1H, t, J ) 7 Hz), 7.72 (1H, d, J ) 8.5 Hz); IR (KBr) 2641, 2566, 1703, 1675 cm-1. Anal. (C22H20N2O6‚1/4H2O) C,H,N. Similarly, 25b-g, 26a-e, 33a-c, 34a-c, 37, and 38 were prepared from the corresponding ethyl esters. 25b: 88%; mp 195 °C. Anal. (C23H24N2O5) C,H,N. 25c: 91%; mp 180-182 °C (EtOH). Anal. (C24H26N2O5‚1/2H2O) C,H,N. 25d: 81%; mp 190-192 °C dec (EtOH). Anal. (C25H28N2O5) C,H,N. 25e: quantitative; mp 222-224 °C. Anal. (C24H26N2O4) C,H,N. 25f: 77%; mp 181-182 °C (MeOH). Anal. (C25H28N2O5) C,H,N. 25g: 95%; mp 155-158 °C (EtOH-EtOAc). Anal. (C26H30N2O5‚H2O) C,H,N. 26a: 76%; mp 229.5-232 °C (MeOH). Anal. (C22H20N6O4) C,H,N. 26b: 96%; mp 184-187 °C (soften), 188-189 °C (melt). Anal. (C23H24N6O3‚H2O) C,H,N. 26c: 94%; mp 199-201 °C (EtOH). Anal. (C24H26N6O3) C,H,N. 26d: 56%; mp 178-181 °C (EtOH). Anal. (C25H28N6O3‚ 1/ C H OH) C,H,N. 26e: 89%; mp 167 °C (soften). Anal. 2 2 5 (C24H26N6O2‚1/4H2O) C,H,N. 33a: 72%; mp 226-228 °C dec (EtOH). Anal. (C21H20N2O4) C,H,N. 33b: 94%; mp 206-207 °C. Anal. (C24H24N2O4) C,H,N. 33c: 85%; mp 165-169 °C (MeOH-Et2O). Anal. (C23H24N2O5‚1/2H2O) C,H,N. 34a: 87%; mp 232-233 °C dec. Anal. (C21H20N6O2) C,H,N. 34b: 85%; mp 179-182 °C (soften), 184 °C (melt). Anal. (C24H24N6O2‚1/ 3H2O) C,H,N. 34c: 77%; mp 229-232 °C (MeOH). Anal. (C23H24N6O3) C,H,N. 37: 76%; mp 173-175 °C dec (EtOHEtOAc). Anal. (C21H20N2O4‚1/2C2H5OH) C,H,N. 38: 62%; mp 208-210 °C dec. Anal. (C21H20N6O2‚1/2C2H5OH) C,H,N. Ethyl 1-[[2′-(tert-Butoxycarbonyl)biphenyl-4-yl]methyl]4-(hydroxymethyl)-2-propylimidazole-5-carboxylate (39). To a solution of 21a (1.94 g, 3.73 mmol) in THF (15 mL) was added a 1.5 M solution of diisobutylaluminum hydride in toluene (5.22 mL, 7.83 mmol) at -30 to -20 °C under N2 atmosphere. The mixture was allowed to stand at -5 °C for 20 h. After adding aqueous NH4Cl and EtOAc, the mixture was stirred at room temperature for 2 h. The precipitates were

334


filtered off through a Celite pad. The organic phase was separated, dried over MgSO4, and concentrated in vacuo. The residue was purified by flash column chromatography (MeOH/ CH2Cl2, 1:15) to give crystalline 39 which was washed with IPE: yield 1.47 g (82%); mp 113 °C (EtOAc-IPE); NMR (CDCl3) 0.99 (3H, t, J ) 7.5 Hz), 1.24 (9H, s), 1.31 (3H, t, J ) 7 Hz), 1.80 (2H, sx, J ) 7.5 Hz), 2.69 (2H, t, J ) 7.5 Hz), 3.63 (1H, br s), 4.27 (2H, q, J ) 7 Hz), 4.88 (2H, s), 5.61 (2H, s), 7.00 (2H, d, J ) 8 Hz), 7.27 (2H, d, J ) 8 Hz), 7.27 (1H, d,d, J ) 1, 7 Hz), 7.39 (1H, d,t, J ) 1, 7.5 Hz), 7.48 (1H, d,t, J ) 1, 7.5 Hz), 7.77 (1H, d,d, J ) 1, 7.5 Hz); IR (KBr) 3234, 1706, 1698 cm-1. Anal. (C28H34N2O5) C,H,N. Similarly, 40 and 51 were prepared from 22a and 45. 40: 69%; mp 134-136 °C (EtOAc). Anal. (C43H40N6O3) C,H,N. 51: 77%; mp 176-177 °C. Anal. (C26H32N2O4) H,N; C: calcd, 71.53; found, 71.12. Ethyl 1-[(2′-Carboxybiphenyl-4-yl)methyl]-4-(hydroxymethyl)-2-propylimidazole-5-carboxylate (41). By the same procedure as described in the preparation of 23a, 39 (0.84 g, 1.76 mmol) was treated with 4 N HCl in dioxane (10 mL) to give 41 (0.69 g, 93%): mp 224-226 °C (EtOH); NMR (DMSOd6) 0.89 (3H, t, J ) 7.5 Hz), 1.22 (3H, t, J ) 7.5 Hz), 1.64 (2H, sx, J ) 7.5 Hz), 2.62 (2H, t, J ) 7.5 Hz), 4.19 (2H, q, J ) 7.5 Hz), 4.60 (2H, d, J ) 5 Hz), 4.75 (1H, t, J ) 5 Hz), 5.58 (2H, s), 7.01 (2H, d, J ) 8.5 Hz), 7.29 (2H, d, J ) 8.5 Hz), 7.34 (1H, d, J ) 7.5 Hz), 7.44 (1H, t, J ) 7.5 Hz), 7.55 (1H, t, J ) 7.5 Hz), 7.71 (1H, d, J ) 7.5 Hz), 12.74 (1H, s); IR (KBr) 3410, 2452, 1701 cm-1. Anal. (C24H26N2O5) C,H,N. Similarly, 47 and 52 were prepared. 47: quantitative; mp 215 °C (soften), 230 °C (melt) (EtOH). Anal. (C24H26N2O5) C,H,N. 52: obtained as HCl salt; quantitative; mp 200 °C (color), 217 °C (melt). Anal. (C22H24N2O4‚HCl‚1/3H2O) C,H, Cl,N. Ethyl 4-(Hydroxymethyl)-2-propyl-1-[(2′-1H-tetrazol5-ylbiphenyl-4-yl)methyl]imidazole-5-carboxylate (42). By the same procedure as described in the preparation of 24a, 40 (700 mg, 1.02 mmol) was treated with 25% aqueous AcOH to give crystalline 42 (293 mg, 64%): mp 160-162 °C (EtOAcIPE); NMR (DMSO-d6) 0.87 (3H, t, J ) 7.5 Hz), 1.20 (3H, t, J ) 7 Hz), 1.59 (2H, sx, J ) 7.5 Hz), 2.57 (2H, t, J ) 7.5 Hz), 4.17 (2H, q, J ) 7 Hz), 4.58 (2H, s), 4.74 (1H, br s), 5.53 (2H, s), 6.92 (2H, d, J ) 8 Hz), 7.05 (2H, d, J ) 8 Hz), 7.51-7.58 (2H, m), 7.63-7.69 (2H, m); IR (KBr) 1710 cm-1. Anal. (C24H26N6O3) C,H,N. Similarly, 48 was prepared. 48: quantitative; mp 128 °C (soften). Anal. (C24H26N6O3) C,H,N. 1-[(2′-Carboxybiphenyl-4-yl)methyl]-4-(hydroxymethyl)2-propylimidazole-5-carboxylic Acid (43). By the same procedure as described in the preparation of 25a, 41 (584 mg, 1.38 mmol) was hydrolyzed with LiOH‚H2O (232 mg, 5.54 mmol) to give 43 (520 mg, 96%): mp 216-218 °C dec (MeOH); NMR (DMSO-d6) 0.88 (3H, t, J ) 7.5 Hz), 1.62 (2H, sx, J ) 7.5 Hz), 2.57 (2H, t, J ) 7.5 Hz), 4.62 (2H, s), 5.64 (2H, s), 7.04 (2H, d, J ) 8 Hz), 7.29 (2H, d, J ) 8 Hz), 7.35 (1H, d, J ) 7.5 Hz), 7.44 (1H, t, J ) 7.5 Hz), 7.55 (1H, d,t, J ) 1, 7.5 Hz), 7.70 (1H, d, J ) 7.5 Hz); IR (KBr) 2610, 2499, 1717, 1634, 1599 cm-1. Anal. (C22H22N2O5) C,H,N. Similarly, 44, 49, and 50 were obtained by hydrolysis of 42, 47, and 48, respectively. 44: 95%; mp 223-224 °C dec (EtOH). Anal. (C22H22N6O3) C,H,N. 49: quantitative; mp 210-212 °C dec (EtOH). Anal. (C22H22N2O5‚1/2C2H5OH) C,H,N. 50: quantitative; mp >270 °C. Anal. (C22H22N6O3) C,H,N. Ethyl 1-[[2′-(tert-Butoxycarbonyl)biphenyl-4-yl]methyl]5-(hydroxymethyl)-2-propylimidazole-4-carboxylate (45). A mixture of 21a (5.87 g, 11.3 mmol) and LiAlH(OBut)3 (5.73 g, 22.6 mmol) in THF (59 mL) was stirred at room temperature for 5 days. During the above reaction period, two portions of additional LiAl(OBut)3 (1.43 g × 2, 11.3 mmol) were added to the reaction mixture, respectively, on the second and third days. MeOH (10 mL) was added under ice cooling, and the mixture was stirred for 10 min. The solvent was removed by distillation in vacuo, and the residue was dissolved in EtOAc and H2O. After removal of the precipitates by filtration through a Celite pad, the organic phase was separated, dried over Na2SO4, and concentrated in vacuo. The residue was subjected to flash column chromatography (EtOAc/CH2Cl2, 1:1)

Yanagisawa et al. to give the following fractions. Faster moving fraction 45: yield 3.55 g (66%); mp 123-124.5 °C (IPE); NMR (CDCl3) 0.96 (3H, t, J ) 8 Hz), 1.26 (9H, s), 1.43 (3H, t, J ) 7 Hz), 1.73 (2H, sx, J ) 8 Hz), 2.67 (2H, t, J ) 8 Hz), 3.73 (1H, t, J ) 6.5 Hz), 4.44 (2H, q, J ) 7 Hz), 4.72 (2H, d, J ) 6.5 Hz), 5.26 (2H, s), 6.97 (2H, d, J ) 8.5 Hz), 7.26-7.31 (3H, m), 7.42 (1H, t, J ) 7 Hz), 7.49 (1H, t, J ) 7 Hz), 7.78 (1H, d, J ) 6.5 Hz); IR (KBr) 3310, 1715 cm-1. Anal. (C28H34N2O5) C,H,N. Slower moving fraction 39 was obtained as a mixture of 39 and 45: yield 0.45 g (10%). Similarly, 22a was reduced with LiAlH(OBut)3 to give 46 and 40. 46: 79%; mp 162-164 °C (color), 174-177 °C (melt). Anal. (C43H40N6O3) C,H,N. 40: 8.3% (as a mixture of 22a and 40). Ethyl 1-[[2′-(tert-Butoxycarbonyl)biphenyl-4-yl]methyl]4-methyl-2-propylimidazole-5-carboxylate (53a). To a solution of 39 (479 mg, 1.00 mmol) and Et3N (0.35 mL, 2.52 mmol) in CH2Cl2 (6 mL) was added ClCOCO2Et (165 mg, 1.21 mmol) under ice cooling and N2. After stirring at room temperature for 0.5 h, the reaction mixture was diluted with EtOAc and washed with aqueous NaHCO3. The organic phase was dried over MgSO4 and concentrated in vacuo to give the 4-ethoxyglyoxylyl ester of 39 as a syrup (590 mg, quantitative): NMR (CDCl3) 0.98 (3H, t, J ) 7.5 Hz), 1.23 (9H, s), 1.29 (3H, t, J ) 7.5 Hz), 1.36 (3H, t, J ) 7 Hz), 1.78 (2H, sx, J ) 7.5 Hz), 2.67 (2H, t, J ) 7.5 Hz), 4.27 (2H, q, J ) 7 Hz), 4.35 (2H, q, J ) 7.5 Hz), 5.53 (2H, s), 5.62 (2H, s), 7.02 (2H, d, J ) 8 Hz), 7.25-7.29 (3H, m), 7.39 (1H, t, J ) 7.5 Hz), 7.48 (1H, t, J ) 7.5 Hz), 7.77 (1H, d, J ) 7.5 Hz). To a solution of the above ester (544 mg, 0.92 mmol) in toluene (10 mL) were added Bu3SnH (600 mg, 2.06 mmol) and 2,2′-azobis(isobutyronitrile) (20 mg, 0.12 mmol), and the mixture was refluxed for 1.5 h under N2. The reaction solution was concentrated in vacuo. The residue was purified by flash column chromatography (EtOAc/hexane, 1:1) to give 53a as a syrup (282 mg, 61%): NMR (CDCl3) 0.97 (3H, t, J ) 7.5 Hz), 1.22 (9H, s), 1.31 (3H, t, J ) 7.5 Hz), 1.76 (2H, sx, J ) 7.5 Hz), 2.52 (3H, s), 2.63 (2H, t, J ) 7.5 Hz), 4.24 (2H, q, J ) 7.5 Hz), 5.58 (2H, s), 6.99 (2H, d, J ) 8 Hz), 7.25 (2H, d, J ) 8 Hz), 7.28 (1H, d, J ) 8 Hz), 7.38 (1H, t, J ) 8 Hz), 7.47 (1H, t, J ) 8 Hz), 7.76 (1H, d, J ) 8 Hz); IR (liquid film) 1703 cm-1. Anal. (C28H34N2O4) C,H,N. Similarly, 53b and 54a,b were prepared from 27c, 40, and 29c, respectively. 53b: 45%; syrup. Anal. (C29H36N2O4) C,H,N. 54a: 87%; mp 178.5-179 °C dec (MeCN-EtOAc). Anal. (C43H40N6O2) C,H,N. 54b: 52%; mp 139-141 °C (IPEhexane). Anal. (C44H42N6O2) C,H,N. Ethyl 1-[(2′-Carboxybiphenyl-4-yl)methyl]-4-methyl-2propylimidazole-5-carboxylate (55a). By the same procedure as described in the preparation of 23a, 53a (1.30 g, 2.80 mmol) was treated with 4 N HCl in dioxane to give 55a (1.02 g, 89%): mp 186-188 °C (EtOH-EtOAc); NMR (DMSO-d6) 0.87 (3H, t, J ) 7.5 Hz), 1.21 (3H, t, J ) 7 Hz), 1.62 (2H, sx, J ) 7.5 Hz), 2.39 (3H, s), 2.58 (2H, t, J ) 7.5 Hz), 4.18 (2H, q, J ) 7 Hz), 5.56 (2H, s), 6.99 (2H, d, J ) 8 Hz), 7.28 (2H, d, J ) 8 Hz), 7.34 (1H, d,d, J ) 1, 7.5 Hz), 7.44 (1H, d,t, J ) 1, 7.5 Hz), 7.55 (1H, d,t, J ) 1, 7.5 Hz), 7.71 (1H, d,d, J ) 1, 7.5 Hz), 12.75 (1H, br s); IR (KBr) 2438, 1704 cm-1. Anal. (C24H26N2O4) C,H,N. Similarly, 55b was prepared. 55b: 89%; mp 181-182 °C (EtOAc). Anal. (C25H28N2O4) C,H,N. Ethyl 4-Methyl-2-propyl-1-[(2′-1H-tetrazol-5-ylbiphenyl-4-yl)methyl]imidazole-5-carboxylate (56a). By the same procedure as described in the preparation of 24a, 54a (1.15 g, 1.71 mmol) was treated with 25% aqueous AcOH to give 56a (710 mg, 96%): mp 179.5-180.5 °C (EtOAc); NMR (CDCl3) 0.84 (3H, t, J ) 7.5 Hz), 1.18 (3H, t, J ) 7.5 Hz), 1.54 (2H, sx, J ) 7.5 Hz), 1.93 (3H, s), 2.18 (2H, t, J ) 7.5 Hz), 4.14 (2H, q, J ) 7.5 Hz), 5.40 (2H, s), 6.70 (2H, d, J ) 8 Hz), 7.03 (2H, d, J ) 8 Hz), 7.44 (1H, d,d, J ) 1.5, 7.5 Hz), 7.52-7.62 (2H, m), 7.87 (1H, d,d, J ) 1.5, 7.5 Hz); IR (KBr) 1705 cm-1. Anal. (C24H26N6O2) C,H,N. Similarly, 56b was prepared. 56b: 95%; mp 182-183 °C (EtOAc-Et2O). Anal. (C25H28N6O2) C,H,N. 1-[(2′-Carboxybiphenyl-4-yl)methyl]-4-methyl-2-propylimidazole-5-carboxylic Acid (57a). By a procedure

Imidazolecarboxylic Acids as AII Receptor Antagonists similar to the preparation of 25a, 55a (570 mg, 1.40 mmol) was hydrolyzed with NaOH (304 mg, 7.6 mmol) at 70 °C for 5 h to give 57a (498 mg, 94%): mp 197-199 °C; NMR (DMSOd6) 0.87 (3H, t, J ) 7.5 Hz), 1.61 (2H, sx, J ) 7.5 Hz), 2.41 (3H, s), 2.61 (2H, t, J ) 7.5 Hz), 5.63 (2H, s), 7.03 (2H, d, J ) 8 Hz), 7.29 (2H, d, J ) 8 Hz), 7.35 (1H, d,d, J ) 1, 7.5 Hz), 7.44 (1H, d,t, J ) 1, 7.5 Hz), 7.55 (1H, d,t, J ) 1, 7.5 Hz), 7.71 (1H, d,d, J ) 1, 7.5 Hz), 12.85 (2H, br s); IR (KBr) 1713, 1638 (wk) cm-1. Anal. (C22H22N2O4‚1/2H2O) C,H,N. Similarly, 57b and 58a,b were prepared. 57b: 94%; mp 220-222 °C dec. Anal. (C23H24N2O4) C,H,N. 58a: 97%; mp 202.5-204 °C dec. Anal. (C22H22N6O2) C,H,N. 58b: 98%; mp 198-199 °C dec (EtOH-Et2O). Anal. (C23H24N6O2‚1/3H2O) C,H,N. 1-[[2′-(tert-Butoxycarbonyl)biphenyl-4-yl]methyl]-4-(1hydroxy-1-methylethyl)-2-propylimidazole-5-carboxylic Acid (59). After stirring a mixture of 21c (7.5 g, 14.8 mmol) and LiOH‚H2O (3.10 g, 73.8 mmol) in dioxane (39 mL) and H2O (65 mL) at room temperature for 20 h, dioxane was removed by distillation in vacuo. To the concentrate was dropwise added 1 N HCl (73.6 mL) under ice cooling to give 59 as precipitates, which were collected by filtration and washed with H2O and IPE: yield 7.09 g (quantitative); mp 148-150 °C (EtOAc-IPE); NMR (CDCl3) 0.89 (3H, t, J ) 7.5 Hz), 1.24 (9H, s), 1.62 (2H, sx, J ) 7.5 Hz), 1.69 (6H, s), 2.96 (2H, t, J ) 7.5 Hz), 5.88 (2H, s), 7.09 (2H, d, J ) 8 Hz), 7.217.26 (3H, m), 7.38 (1H, d,t, J ) 1, 7.5 Hz), 7.45 (1H, d,t, J ) 1, 7.5 Hz), 7.76 (1H, d,d, J ) 1, 7.5 Hz); IR (KBr) 2498, 1708, 1625, 1590 cm-1. Anal. (C28H34N2O5) C,H,N. (5-Methyl-2-oxo-1,3-dioxol-4-yl)methyl 1-[[2′-(tert-Butoxycarbonyl)biphenyl-4-yl]methyl]-4-(1-hydroxy-1methylethyl)-2-propylimidazole-5-carboxylate (61a). To a mixture of 59 (1.00 g, 2.09 mmol) and K2CO3 (0.60 g, 4.34 mmol) in DMA (10 mL) was added a solution of 4-(chloromethyl)-5-methyl-2-oxo-1,3-dioxole (74% purity, 621 mg, 3.10 mmol) in DMA (6 mL) dropwise. The mixture was stirred at room temperature for 1 h and then at 50 °C for 2 h. EtOAc and H2O were added. The organic phase was separated, washed with brine, dried over anhydrous MgSO4, and concentrated in vacuo to give crystalline 61a (964 mg, 78%) which was washed with EtOAc-hexane (1:1). The mother solution was concentrated in vacuo, and the residue was subjected to flash column chromatography (EtOAc/hexane, 1:1) to give further 61a (140 mg, 11%): 152-155 °C (EtOAc-IPE); 0.98 (3H, t, J ) 7.5 Hz), 1.27 (9H, s), 1.63 (6H, s), 1.76 (2H, sx, J ) 7.5 Hz), 2.07 (3H, s), 2.68 (2H, t, J ) 7.5 Hz), 4.89 (2H, s), 5.46 (2H, s), 5.57 (1H, s), 6.90 (2H, d, J ) 8 Hz), 7.28 (2H, d, J ) 8 Hz), 7.32 (1H, d, J ) 7.5 Hz), 7.39 (1H, t, J ) 7.5 Hz), 7.50 (1H, t, J ) 7.5 Hz), 7.76 (1H, d, J ) 7.5 Hz); IR (KBr) 3435, 1828, 1742 (sh), 1705, 1673 cm-1. Anal. (C33H38N2O8) C,H,N. Similarly, 61b-d and 65 were prepared. 61b: 98%; mp 104-106 °C (IPE). Anal. (C34H44N2O7) C,H,N. 61c: 85%; mp 89-92 °C (IPE-hexane). Anal. (C33H42N2O8) C,H,N. 61d: 95%; mp 143-146 °C (EtOAc-IPE). Anal. (C36H38N2O7) C,H,N. 65: 96%; mp 97-98 °C (IPE). Anal. (C35H40N2O5) C,H,N. (5-Methyl-2-oxo-1,3-dioxol-4-yl)methyl 1-[(2′-Carboxybiphenyl-4-yl)methyl]-4-(1-hydroxy-1-methylethyl)-2-propylimidazole-5-carboxylate (63a). By the same procedure as described in the preparation of 23a, 61a (0.87 g, 1.47 mmol) was treated with 4 N HCl in dioxane to give 63a (0.79 g, quantitative): mp 171-172 °C (EtOAc-IPE); NMR (DMSOd6) 0.91 (3H, t, J ) 7.5 Hz), 1.49 (6H, s), 1.64 (2H, sx, J ) 7.5 Hz), 2.08 (3H, s), 2.65 (2H, t, J ) 7.5 Hz), 5.07 (2H, s), 5.23 (1H, s), 5.47 (2H, s), 6.96 (2H, d, J ) 8 Hz), 7.27 (2H, d, J ) 8 Hz), 7.36 (1H, d, J ) 7.5 Hz), 7.44 (1H, t, J ) 7.5 Hz), 7.56 (1H, t, J ) 7.5 Hz), 7.71 (1H, d, J ) 7.5 Hz), 12.73 (1H, br s); IR (KBr) 3431, 2613, 2501, 1825, 1735 (sh), 1711 (sh), 1687 cm-1. Anal. (C29H30N2O8) C,H,N. Similarly, 63b-d and 66 were prepared. 63b: quantitative; mp 156 °C (EtOAc-IPE). Anal. (C30H36N2O7) C,H,N. 63c: 95%; mp 147-149 °C (EtOAc-IPE). Anal. (C29H34N2O8) C,H,N. 63d: quantitative; mp 187-188 °C (EtOAc-IPE). Anal. (C32H30N2O7) C,H,N. 66: quantitative; mp 147-149 °C (EtOAc-IPE). Anal. (C31H32N2O5) C,H,N.

Journal of Medicinal Chemistry, 1996, Vol. 39, No. 1 335 (5-Methyl-2-oxo-1,3-dioxol-4-yl)methyl 4-(1-Hydroxy-1methylethyl)-2-propyl-1-[[2′-[2-(triphenylmethyl)-2H-tetrazol-5-yl]biphenyl-4-yl]methyl]imidazole-5-carboxylate (62a). After stirring a mixture of 22c (30.0 g, 41.8 mmol) and LiOH‚H2O (2.65 g, 63.1 mmol) in dioxane (344 mL) and H2O (158 mL) at 5-10 °C for 20 h, the reaction solution was concentrated in vacuo to a volume of about 100 mL. EtOAc (0.6 L) and NaCl were added. The organic phase was separated, dried over Na2SO4, and concentrated in vacuo. The residual gum, the lithium salt of 60, was dried at 50 °C in vacuo and then dissolved in DMA (160 mL). To the solution were added powdered K2CO3 (6.08 g, 44 mmol) and a solution of 4-(chloromethyl)-5-methyl-2-oxo-1,3-dioxole (74% purity, 11.2 g, 55.8 mmol) in DMA (16 mL) dropwise, successively. The mixture was stirred at room temperature for 1 h and at 50 °C for 3 h. EtOAc and H2O were added. The organic phase was separated, dried over MgSO4, and concentrated in vacuo. The residual crystals were washed with EtOAc-IPE to give 62a (29.3 g, 88%): mp 102-104 °C (EtOAc-IPE); NMR (CDCl3) 0.89 (3H, t, J ) 7.5 Hz), 1.62 (6H, s), 1.60-1.75 (2H, m), 1.97 (3H, s), 2.54 (2H, t, J ) 8 Hz), 4.70 (2H, s), 5.30 (2H, s), 5.61 (1H, s), 6.68 (2H, d, J ) 7.5 Hz), 6.90-7.52 (20H, m), 7.87 (1H, d, J ) 7.5 Hz); IR (KBr) 3420, 1825, 1738 (wk), 1707, 1678 cm-1. Anal. (C48H44N6O6) C,H,N. Similarly, 62b was prepared. 62b: 96%; amorphous powder. Anal. (C49H50N6O5) C,H,N. (5-Methyl-2-oxo-1,3-dioxol-4-yl)methyl 4-(1-Hydroxy-1methylethyl)-2-propyl-1-[(2′-1H-tetrazol-5-ylbiphenyl-4yl)methyl]imidazole-5-carboxylate (64a) (CS-866). By the same procedure as described in the preparation of 24a, 62a (29.3 g, 36.6 mmol) was treated with 25% aqueous AcOH to afford 64a (16.6 g, 81%): mp 180-182 °C dec (EtOH); NMR (DMSO-d6) 0.88 (3H, t, J ) 7.5 Hz), 1.47 (6H, s), 1.58 (2H, sx, J ) 7.5 Hz), 2.08 (3H, s), 2.60 (2H, t, J ) 7.5 Hz), 5.05 (2H, s), 5.20 (1H, s), 5.42 (2H, s), 6.86 (2H, d, J ) 8.5 Hz), 7.04 (2H, d, J ) 8.5 Hz), 7.52-7.70 (4H, m); IR (KBr) 3291, 1833, 1740 (sh), 1708 cm-1. Anal. (C29H30N6O6) C,H,N. Similarly, 64b was prepared. 64b: 89%; amorphous powder. Anal. (C30H36N6O5) C,H,N. Benzyl 4-(1-Hydroxy-1-methylethyl)-1-[[2′-[[(pivaloyloxy)methoxy]carbonyl]biphenyl-4-yl]methyl]-2-propylimidazole-5-carboxylate (67). By the same procedure as the preparation of 61a, 66 (1.07 g, 2.08 mmol) was esterified with ClCH2OCOBut (0.47 g, 3.12 mmol) to give 67 (1.30 g, quantitative) as a syrup: NMR (CDCl3) 0.96 (3H, t, J ) 7.5 Hz), 1.19 (9H, s), 1.62 (6H, s), 1.72 (2H, sx, J ) 7.5 Hz), 2.65 (2H, t, J ) 7.5 Hz), 5.17 (2H, s), 5.40 (2H, s), 5.78 (2H, s), 5.79 (1H, s), 6.77 (2H, d, J ) 8 Hz), 7.19-7.24 (3H, m), 7.28 (2H, d, J ) 8 Hz), 7.25-7.33 (2H, m), 7.33 (1H, d, J ) 7.5 Hz), 7.43 (1H, t, J ) 7.5 Hz), 7.58 (1H, t, J ) 7.5 Hz), 7.89 (1H, d, J ) 7.5 Hz); IR (liquid film) 3437, 1754, 1705, 1603 cm-1. Anal. (C37H42N2O7) C,H,N. 4-(1-Hydroxy-1-methylethyl)-1-[[2′-[[(pivaloyloxy)methoxy]carbonyl]biphenyl-4-yl]methyl]-2-propylimidazole-5-carboxylic Acid (68). A mixture of 67 (970 mg, 1.55 mmol) and 5% Pd-C (100 mg) in MeOH (15 mL) was stirred under a hydrogen atmosphere at room temperature for 3 h. The catalyst was removed by filtration, and the filtrate was concentrated in vacuo to give a syrup which was crystallized in EtOAc-IPE: yield 0.64 g (77%); mp 146-148 °C (EtOAcIPE); NMR (DMSO-d6) 0.88 (3H, t, J ) 7.5 Hz), 1.12 (9H, s), 1.53 (6H, s), 1.59 (2H, sx, J ) 7.5 Hz), 2.60 (2H, t, J ) 7.5 Hz), 5.70 (2H, s), 5.73 (2H, s), 7.02 (2H, d, J ) 8.5 Hz), 7.25 (2H, d, J ) 8.5 Hz), 7.43 (1H, d,d, J ) 1, 7.5 Hz), 7.50 (1H, d,t, J ) 1, 7.5 Hz), 7.65 (1H, d,t, J ) 1, 7.5 Hz), 7.74 (1H, d,d, J ) 1, 7.5 Hz); IR (KBr) 3426, 3211, 2623, 1753, 1633, 1597 cm-1. Anal. (C30H36N2O7) C,H,N. AII Receptor (AT1) Binding Assay. Membrane fractions of bovine adrenal cortex were prepared by modifications of the method of Maeda et al.41 The freshly isolated bovine adrenal cortex was homogenized in ice-cold medium containing 10 mM sodium phosphate buffer (pH 7.4), 30 mM NaCl, 1 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol (DTT), 1 µM (p-amidinophenyl)methanesulfonyl fluoride HCl (p-APMSF), and 0.02% NaN3. The homogenate was layered on a 41% sucrose solution and centrifuged at 95000g for 60 min. The interfacial band

336


Yanagisawa et al.

Table 4. Experimental Data and Final Refinement Results for 26c, 39, and 64a molecular formula formula weight space group crystal system a (Å) b (Å) c (Å) β (deg) V (Å3) Z Dcalcd (g cm-3) radiation wave length (Å) crystal size (mm) F(000) 2θmax (deg) reflections measured reflections Fo g 3σFo weighting scheme, w final R final Rw Fourier excursions (eÅ-3)

26c

39

64a

C24H25N6O3 446.51 P21/c monoclinic 10.067 (3) 17.992 (2) 14.214 (5) 91.39 (5) 2573.6 (5) 4 1.15 Cu KR 1.541 78 0.3 × 0.3 × 0.3 944 128 4311 2976 1/[1 + [(Fo - 35)/45]2] 0.045 0.043 0.10, -0.14

C28H34N2O5 478.59 P21/n monoclinic 19.756 (2) 16.569 (2) 8.200 (1) 101.31 (1) 2632.1 (8) 4 1.21 Cu KR 1.541 78 0.3 × 0.3 × 0.2 1024 128 4098 2144 1/[1 + [(Fo - 15/70]2] 0.064 0.063 0.6, -0.16

C29H30N6O6 558.59 P21/c monoclinic 12.404 (2) 21.256 (2) 10.969 (1) 101.56 (1) 2833.1 (5) 4 1.31 Cu KR 1.541 78 0.4 × 0.3 × 0.3 1176 128 4776 3543 1.0 0.061 0.059 0.20, -0.28

between the supernatant and the sucrose portion was collected. The membrane fraction was washed by centrifugation at 95000g for 20 min. The pellet obtained was used as the source of AII receptor. Binding of [125I]AII to membranes was performed at 22 °C for 120 min in 96-well plates. Each 200 µL incubated solution contained the following (final concentration): 20 mM Tris HCl (pH 7.4), 120 mM NaCl, 5 mM MgCl2, 0.05% bovine serum albumin (BSA), 1 µM p-APMSF, 0.5 mM EDTA, 0.1 mM DTT, 0.1 nM [125I]AII (specific activity 2200 Ci/mmol), the test compound (appropriate concentration), and membrane preparations (10 µg of protein/well). At the end of the incubation, bound complex was trapped on filters (GF/C) and washed with cold Tris buffer (pH 7.4; 3 × 250 µL). Filter disks were dried, punched out, and counted in a γ-counter. Specific binding was defined as total binding minus nonspecific binding, which was estimated in the presence of 1 µM unlabeled AII. The IC50 of an inhibitor was determined as the concentration that displaced the specifically bound [125I]AII by 50%. Antagonism of AII-Induced Pressor Response by Intravenous Administration. Male Wistar-Imamichi rats weighing 300-400 g were anesthetized with inactin (100 mg/ kg, ip). An animal was surgically prepared with an aortic cannula inserted via the left femoral artery for measuring blood pressure and with a caval cannula inserted via the left femoral vein for injecting AII and a test compound. The aortic cannula was connected to a pressure transducer (TP-200T, Nihon Kohden), and mean blood pressure and heart rate were continuously recorded on a pen-writing recorder (WT-685G, Nihon Kohden). AII (50 ng/kg) was intravenously administered via the cannula at intervals of 10 min, and the pressor response (about 50 mmHg) was observed. After constant pressor responses to AII were obtained, a test compound was intravenously administered. Two minutes later, AII (50 ng/ kg) was again injected, and the inhibitory effect of the test compound was estimated. The percent inhibitions of the pressor response to AII with progressive increases of the test compound were used to calculate the value of ID50, which was determined as the dose that inhibited the AII-induced pressor response by 50%. AII and the test compound were dissolved in 0.5% BSA and DMSO, respectively. Antagonism of AII-Induced Pressor Response by Oral Administration. Male Wistar-Imamichi rats weighing 300400 g were anesthetized with sodium pentobarbital (50 mg/ kg, ip), and two cannulae were placed by the same procedure described above. The other end of the venous cannula was led under the skin and exteriorized at the back of the neck. A rat was placed in an individual cage after surgery and fasted for 24 h. On the next day, the aortic cannula was connected to a pressure transducer (TP-200T, Nihon Kohden), and mean blood pressure and heart rate were continuously recorded on

a pen-writing recorder (WT-685G, Nihon Kohden). After blood pressure and heart rate were stabilized, AII was administered at a dose of 50 ng/kg via the venous cannula. Intravenous administration of AII was repeated until a constant response was obtained, and then the test compound was orally administered. Intravenous administrations of AII (50 ng/kg) were repeated at appropriate intervals for 8 h. The pressor responses were recorded, and the inhibitory effect of the test compound was expressed as the percentage of the inhibition of the AII pressor response. AII and the test compound were dissolved in 0.5% BSA and 50% aqueous DMF, respectively. X-ray Structural Analysis of 26c, 39, and 64a. Compounds 26c, 39, and 64a were crystallized from EtOH, IPE, and EtOH diffusion systems, respectively. Lattice constants were determined by least-squares fit of angular settings of 20 reflections with the range 20° < 2θ < 50°. Intensity data were obtained on a Rigaku AFC-5R diffractometer, using the ω-2θ scan technique. All intensities were corrected for Lorentz and polarization effects but not for absorption. Three structures of 26c, 39, and 64a were solved by the direct methods using MULTAN 7842 and refined by the block-diagonal least-squares technique with anisotropic thermal parameters. Hydrogen atoms were located on a difference Fourier synthesis and refined isotropically. A difference Fourier map revealed two positions of the terminal methyl group of the propyl group of 39, which were included with population parameters of 0.75 and 0.25. Atomic scattering factors were taken from the Revised and Supplementary Tables.43 Calculations were carried out with the DIRECT-SEARCH program system.44 The experimental data and final refinement results for 26c, 39, and 64a are presented in Table 4. Full crystallographic details are available as supporting information. Molecular Modeling. All calculations were performed on a microVAX 4200 and an IRIS workstation. Geometry optimization of 71 in the form of a carboxylate anion, a protonated imidazole ring, and a tetrazole anion was carried out using the PM3 method of the MOPAC program. The torsional angles of τ1-τ8 were incremented by 30°, 120°, 30°, 120°, 30°, 30°, 30°, and 30°, respectively, in the generation of initial conformations. Conformational analysis of 74 in the carboxylate anion form of the C-terminal was performed with the QUANTA/ CHARMm program. Stable conformations of 74 were searched using the random perturbation of 17 rotatable bonds with the window of 120° starting from the two kinds of initial conformations. Perturbed conformations with lower energies

Nonpeptide Angiotensin II Receptor Antagonists: Synthesis, Biological

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